This application is a National Stage Application of International Application Serial No. PCT/EP99/00417, filed on Jan. 21, 1999, and claims priority under 35 U.S.C. xc2xa7119 to GB 9803628.8, filed on Feb. 20, 1998, and GB 9809767.8, filed on May 7, 1998.
The present invention relates to microorganisms producing a taxane, to the use of said microorganisms for the production of a taxane, and to the procedure for the isolation of said microorganisms from plants.
Paclitaxel, a diterpene which is of the chemical structural formula: 
shows significant properties of promoting the polymerization of tubulin and inhibiting the depolymerization of microtubules. For these reasons, paclitaxel is a valuable antileukemic and antitumor agent and is the subject of increasing research. Paclitaxel has been identified in 1971 (Wani M C et al, J. Amer. Chem. Soc. 1971, 93, 2325-2327) by isolating it from the bark of Taxus brevifolia (Pacific yew). The low yield of the isolation of paclitaxel (around 0.007%) from this source and the concerns about the survival of Taxus brevifolia, which is a rather slow growing plant, raised by its massive collection have pushed the research for alternative paclitaxel production methods.
Isolation of paclitaxel from a renewable source such as the needles and the twigs of Taxus species has proven possible although with a yield comparable, if not lower, than that obtainable from the bark of Taxus brevifolia. 
The total chemical synthesis of paclitaxel has been described by Nicolau et al., Nature 1994, 367, 630-634, nevertheless the complexity and low yield of this method have prevented its industrial scale-up.
Semisynthesis of paclitaxel from baccatin III or 10-deacetylbaccatin III, has proved to be a much better option because of the use of a renewable source such as the needles of the European yew Taxus baccata to obtain the precursors.
Several documents on tissue culture of plants of the genus Taxus have been published in recent years, however this procedure is limited by the intrinsic technical difficulties of cell culture on an industrial scale.
Recently, patents and patent applications for the production of paclitaxel from the fermentation of fungi and bacteria isolated from samples of Taxus have been published (WO 93/21338, U.S. Pat. No. 5,322,779, WO 95/04154, U.S. Pat. No. 5,561,055 and WO 96/32490). These confirm that there is a need for improved methods for the production of paclitaxel on industrial scale.
In this specification the used classification rules are described in Bergey""s Manual of Systematic Bacteriology, vol. 4, 1989.
We have surprisingly found a new taxanes producing microorganism of the Actinomycetes group, which is particularly well suited for an industrial fermentation process for the production of a taxane such as paclitaxel and related taxanes. For instance, compared to the fungi of the prior art, the microorganisms of the present invention can be more easily genetically modified to enhance taxane production and have a shorter fermentation time.
The present invention relates to microorganisms of the Actinomycetes group which produce a taxane, such as paclitaxel or related taxanes, as well as to the biologically pure cultures of a strain of the Actinomycetes group which produce a taxane such as paclitaxel or related taxanes.
The microorganisms of the invention preferably belong to the suprageneric groups of Streptomycetes, Actinoplanetes, Maduromycetes, Thermomonosporas or Nocardioforms, more preferably they belong to the genus Streptomyces, Actinoplanes, Nocardiopsis, Micromonospora, Actinomadura or Kitasatosporia and still more preferably to the genus Kitasatosporia.
A preferred strain according to the present invention is a Kitasatosporia sp. deposited under the Budapest treaty at the Colleccion Espaxc3x1ola De Cultivos Tipo, (Biological Science Faculty, University of Valencia, 46100 Burjasot (Valencia), Spain) on Jan. 19, 1998 under the deposit number CECT 4991, and it was isolated from a Taxus baccata sample and proved to produce paclitaxel or other related taxanes. Therefore, the present invention more preferably relates to the biologically pure cultures of the genus Kitasatosporia having all the identifiying characteristics of Kitasatosporia sp. CECT 4991.
Moreover, the present invention provides a procedure for the isolation of microorganisms of the Actinomycetes group which produce a taxane, such as paclitaxel or related taxanes, from plants, including plants of the Taxus genus and related genera, collected from various geographical area.
Preferably, the isolation procedure of microorganisms according to the present invention is carried out on plants selected from plants of Taxus genus or related genera, more preferably Taxus baccata, Taxus brevifolia, or Torreja californica. 
According to the present invention, the procedure for the isolation of microorganisms of the Actinomycetes group producing a taxane, such as paclitaxel or related taxanes, may be carried out, for example, by sterilizing each part of the plant (roots, bark, twigs, needles, stems) with a sterilizing agent, preferably selected from sodium hypochlorite, propylenoxid, a solution of dodecylcarbamylmethyl-benzyldimethylammonium cloride, triethilenglycol sodium nitrite and UV light, by cutting the different parts of the plant in small fragments with a sterile blade and by placing these small fragments on agar culture media in Petri plates or, preferably, by homogenizing the sterilized parts as above, with a blender at room temperature; the resultant homogenate, reduced to powder may be then transferred on agar culture media in Petri plates using a modified Andersen""s sampler. The microorganism colonies grown on the agar plates are then removed and transferred to plates containing water agar media until a pure culture is obtained.
The colonies were grown in liquid media suitable for growth and taxane production, such as conditions suitable for paclitaxel production. The extracts and the fractions obtained from the production cultures can be tested to determine the presence of taxanes, such as paclitaxel or related taxanes, using suitable assays.
In the more preferred procedure for the isolation of the microorganisms of the Actinomycetes group from Taxus and related plants, the powdered samples obtained as above described are introduced into a modified Andersen""s sampler, an apparatus used to analyze the microorganisms population in air samples. Andersen""s sampler is described in detail in: A. Andersen, Journal of Bacteriology 1958, 76 (5), 471-484. This sampler is modified by associating it with a sedimentation chamber in which the powdered samples are introduced. The sedimentation chamber allows isolation of microorganisms present in low percentage from soil and vegetable powdered samples. It consists of a cylindric box made of plexiglass connected to the sampler by a rubber tubing. Plates containing different isolation media were placed in the sampler and the powdered samples were drawn. Depending on their aerodynamic dimensions, the microorganisms present in the powdered samples were collected on plates containing different isolation media. The plates are then removed, incubated and examined. The colonies of microorganisms of the Actinomycetes group grown on the agar plates were picked up and transferred on water agar media in Petri plates until a pure culture was obtained. This method enables the isolation of microorganisms of the Actinomycetes group well separated from fungi and other bacteria.
A further object of the invention is the use of said microorganisms of the Actinomycetes group for the production of a taxane, such as paclitaxel or related taxanes, which comprises the steps of
a) culturing a microorganism of the Actinomycetes group producing the taxanes in a liquid media suitable for its growth and production
b) recovering the taxanes produced.
Thus the invention provides a biologically pure culture of a microorganism of the invention. Such a culture may be substantially free of other microorganisms. The term xe2x80x9crelated taxanesxe2x80x9d is intended to include paclitaxel and all taxane skeleton-containing compounds, which includes taxane skeleton-containing compounds thought to be precursors in the biosynthesis, for example baccatin III, 10-deacetyl baccatin III, cephalomannine, 10-daecetylcephalomannine. The term also includes deacetyltaxol, 7-xylosyltaxol, 7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol and 7-epi-10-deacetylbaccatin III.
The term xe2x80x9crelated taxanesxe2x80x9d also includes taxanes which are recognised by an antibody produced to paclitaxel, 10-deacetyltaxol, 7-xylosyltaxol, 7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol, 7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III, cephalomannine or 10-deacetylcephalornannine. Such antibodies can be polyclonal antibodies or monoclonal antibodies. Polyclonal antibodies can be produced using standard techniques, such as immunising a host animal with paclitaxel, 10-deacetyltaxol, 7-xylosyltaxol, 7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol, 7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III, cephalomannine or 10-deacetylcephalomannine and obtaining the polyclonal antibodies from the animal, for example from the serum. Monoclonal antibodies can also be produced by standard techniques, such as the method of producing monoclonal antibodies of Kohler and Milstein (Nature 256, 495-497 (1975)). To produce such antibodies using the Kohler and Milstein method paclitaxel, 10 deacetyltaxol, 7-xylosyltaxol, 7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol, 7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III, cephalomannine or 10-deacetylcephalomannine would be used to immunise the animal. In particular, xe2x80x9crelated taxanesxe2x80x9d includes those recognised by the antibody R4, 8A10, 3C6 or 3H5. These antibodies are available in kits from Hawaii Biotechnology Group Inc. Preferred xe2x80x9crelated taxanesxe2x80x9d are those which have an IC50 (concentration of taxane required to inhibit the binding of the antibody to solid phase antigen, which may be paclitaxel or baccatin III, by 50% in a competitive immunoassay) of less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, or less than 10 nM, as measured using a competitive immunoassay kit, such as from Hawaii Biotechnology Group Inc.
To grow the isolated colonies the following culture media can be used: water agar, Czapek agar, ISP medium 2 (International Streptomyces Project, Shirling E. B. and Gottlieb D., Int. J. Syst. Bacteriol. 1966, 16, 313-340) adding antifungal and antibacterial substances. The microorganism colonies grown on the agar plates are removed and transferred to water agar media in Petri plates until a pure culture is obtained. After purification, the colonies of the Actinomycetes group producing taxanes, such as paclitaxel or related taxanes, are transferred into tube of ISP medium 3.
Each isolated microorganism can be stored either in glycerol at xe2x88x9280xc2x0 C. or lyophilized. A suitable medium for culturing on organism of the invention may be one which comprises an assimilable carbon source, an assimilable nitrogen source and inorganic salts.
Each microorganism of the Actinomycetes group producing taxanes, such as paclitaxel and related taxanes, may be cultured into liquid seed media, containing complex carbon and nitrogen sources, in Erlenmyer flasks and incubated on a shaker at temperatures ranging from 22xc2x0 to 30xc2x0 C. and at agitation rates from 150 to 250 rpm for 24-48 hours. At the end of incubation, the seed culture is inoculated into different production media containing complex substances such as carbon sources (starch, dextrin, morsuit), nitrogen sources (corn-steep liquor, soybean meal, caseine, soytone, yeast extract), ammonium salts (ammonium sulfate, ammonium chloride) and mineral salts; calcium carbonate and potassium phosphate can be useful. The temperature of incubation can range from 22xc2x0 to 32xc2x0 C. and the agitation rate from 150 to 250 rpm. After inoculation, the fermentation production media are incubated over a period of 2 to 8 days.
After the required incubation time, the cultures are centrifuged and the pellet separated from the supernatant. The supernatant of the culture can be extracted with an organic solvent unmixable with water, for example dichloromethane.
Preferably the supernatant of the culture is loaded onto a pre-packed diatomaceous earth column. This material allows efficient liquid-liquid extraction of lipophilic compounds from aqueous solutions without emulsion problems. Taxanes, such as paclitaxel and other taxanes can be recovered from the column with various organic solvents.
The mycelium cake of the culture can be extracted by homogenizing it with an organic solvent, for example methanol.
Preferably, after homogenizing the mycelium of the culture with a solvent, for example methanol, the pellet is removed by centrifugation, while the solvent extract is dried in vacuo, reconstituted with a small volume of methanol and then with water.
This solution can be loaded onto pre-packed diatomaceous earth columns and treated as the supernatant of the culture.
The fermentation of the isolated microorganisms can be scaled up using fermenter tanks. For instance in order to prepare the inoculum for a 500 L fermenter, the actinomycetes can be cultured in liquid seed media, containing complex substances such as carbon sources (starch, dextrin, morsuit), nitrogen sources (corn-steep liquor, soybean meal, caseine, soytone, yeast extract), ammonium salts (ammonium sulfate, ammonium chloride) and mineral salts; calcium carbonate and potassium phosphate can also be useful.
The temperature of incubation can range from 22xc2x0 to 32xc2x0 C. and the agitation rate from 150 to 250 rpm for 24-48 hours.
At the end of incubation (24 to 48 hours) the seed culture is transferred into an intermediate fermenter containing the same liquid medium as previously described and incubated for 24 to 48 hours at a temperature ranging from 22xc2x0 to 32xc2x0 C., at an agitation rate ranging from 150 to 250 rpm and with an appropriate air flow rate, for instance 68 nL/min for 24-48 hours. Finally a portion of this culture is transferred to the final tank, for example a 500 L fermenter, with a medium containing complex substances such as carbon sources (starch, dextrin, morsuit), nitrogen sources (corn-steep liquor, soybean meal, caseine, soytone, yeast extract), ammonium salts (ammonium sulfate, ammonium chloride) and mineral salts, calcium carbonate or potassium phosphate. After a fermentation period ranging between 96 and 176 hours, the mycelium is removed from the culture by centrifugation and the clear supernatant applied onto a column of chromatographic resin, for instance Amberlite XAD-2.
After the adsorption, the resin can be eluted with solvents, for example ethanol and after removal of the organic solvent under reduced pressure the fractions eluted from the column are extracted with organic solvents unmixable with water such as dichloremethane.
The solvent extract can be purified using reversed-phase C18 columns that are eluted using a step gradient of an alcohol, for example methanol, in water.
A further purification of paclitaxel can be achieved by means of others chromatographic procedures, for example preparative HPLC on RP18 or pentafluorophenyl columns. The extracts and the fractions obtained with the described procedures can be tested to determine the amounts of taxane, such as paclitaxel or related taxanes using suitable assays.
Such an assay may comprise recognition of the taxane by an antibody to a taxane, such as an antibody to paclitaxel, 10 deacetyltaxol, 7-xylosyltaxol, 7-epi-10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, 7-epi-taxol, 7-epi-10-deacetylbaccatin III, baccatin III, 10-deacetyl baccatin III, cephalomannine or 10-deacetylcephalomannine.
Preferably two enzyme immunoassay kits (Indirect Competitive Inhibition Enzyme Immunoassayxe2x80x94CIEIA) employing, respectively, a polyclonal antibody against a taxane skeleton-containing compound and a monoclonal antibody against paclitaxel, are used. Immunoassays and antibodies for paclitaxel can be obtained from Hawaii Biotechnology Group Inc.
Extracts may be analyzed by HPLC, for example on a reversed-phase C18 column using an acetonitrilexe2x80x94water gradient and using a standard curve of paclitaxel concentration versus peak area to quantify paclitaxel in samples.
A further evidence for production of paclitaxel by the isolated strains can be obtained in feeding experiments with radioactive precursors for example with L-[2,3,4,5,6-3H]phenylalanine and 3H-Baccatin and also analytically by liquid chromatography coupled to mass spectroscopy (LC-MS) of the paclitaxel isolated from the cultures. The isolated microorganisms of the Actinomycetes group producing a taxane, such as paclitaxel or related taxanes, can be genetically modified, for example by random mutagenesis or biosynthetic gene manipulation, to enhance taxane production.
The taxane produced by the microorganisms of the invention may act as antitumour agents. A human or animal suffering from a tumor may thus be treated by a method which comprises the administration thereto of an effective amount of such a taxane. The condition of the human or animal may thereby be improved.
Examples of tumors that can be treated are sarcomas, carcinomas, lymphomas, neuroblastomas, melanomas, myelomas, Wilms tumor, leukemias and adenocarcinomas. The taxane can be used to treat ovarian cancer, platinum-resistant ovarian cancer, metastatic breast cancer, non-small cell lung cancer, and head and neck cancer. The taxane may be used to produce a pharmaceutical composition which comprises, as active ingredient, the taxane and a pharmaceutically acceptable carrier or diluent. This composition is usually prepared following conventional methods and is administered in a pharmaceutically suitable form.
Administration can be made by any of the accepted ways for administration of antitumor agents such as intravenous, intramuscular or subcutaneous injection or topical application. For systemic injection the active compound may be, for example, dissolved in a vehicle consisting of a mixture of polyoxyethlated castor oil (Chremophor EL) 50% and ethanol 50% and then diluted with glucose 5% solution at the desired concentration, or in other pharmaceutically suitable carriers.
The amount of the active compound administered depends on the treated subject, age, weight, sex etc., and the severity of the affliction. The method of administration depends on the judgement of the prescribing physician. A suitable dosage for an average 70 kg person may range from about 0.01 g to about 1 g per day.